Fertility determination with transforming growth factor β

ABSTRACT

Therapeutic and diagnostic methods of use for the growth factor transforming growth factor β, are provided by this invention. In accordance with preferred embodiments, methods of determining competence of a conceptus toward uterine implantation are provided as are methods for determining female infertility in mammals.

This is a division of application Ser. No. 08/029,027, filed Mar. 10,1993, now U.S. Pat. No. 5,395,825.

BACKGROUND OF THE INVENTION

In the field of mammalian reproduction, many diagnostic procedures existto aid the reproduction practitioner in making a diagnosis and choosingan appropriate course of action.

Currently, infertility in humans is defined as one year of unprotectedcoitus without conception. Approximately 10-15% of couples are affectedby infertility. The risk of infertility is doubled for women between theages of 35 to 44 as compared to women between the ages of 30 and 34.Approximately 600,000 couples sought professional help during the year1968. However, in the early 1980's this number increased to over 2million visits per year for infertility. Changes in fertility patternswill have a significant impact on the make-up of populations. It hasbeen calculated that by the middle of the next century, the populationin the United States will decline without immigration. Furthermore, thepercent of people over the age of 65 will increase to over 23% in thenext 100 years, resulting in an older and smaller work force.

In the United States, the majority of infertility can be accounted forby problems in the female. Evaluating a female for infertility can becomplex. Examination of the fallopian tubes is an important early stepin mammalian fertility evaluation due to the increased evidence ofpelvic inflammatory disease. Currently, a hysterosalpingogram (HSG) isthe procedure of choice to examine the patency of the fallopian tubes.In addition to HSG, hysteroscopy which is the direct examination of theuterus by a fiber optic device, is important to determine the presenceof endometrial polyps, submucous leiomyomas, and other abnormalitieswithin the uterus itself.

Another category of diagnostic procedures includes examination ofovarian function including ovulation and the secretion of progesteroneduring the luteal phase of the menstrual cycle. Ovarian function can becrudely assessed by measuring basal body temperatures during themenstrual cycle and cervical mucous testing around the time ofovulation. More accurate testing can be performed by measuringluteinizing hormone, a pituitary hormone which induces ovulation after amid-cycle surge. Finally, serum progesterone levels can be measured toassess for normal luteal phase of the menstrual cycle.

The endometrium itself can be directly assessed by performing anendometrial biopsy three days before the suspected onset of menses. Inassessing a mammalian endometrium, current gynecology and infertilityphysicians depend on pathologists to examine endometrial biopsies byhematoxylin and eosin staining of paraffin embedded specimens. Forinfertility patients, the reading of these biopsies provides informationabout the day of the cycle following ovulation, the adequacy of theluteal phase, and other potential data, such as infection, inflammation,or neoplasia of the endometrium. However, in most cases there is noevaluation of the functional and biochemical quality of the endometrium,and often no histologic reading to explain a patient's infertilityproblem.

Finally, the infertility patient can undergo endoscopic examinationthrough an incision in the abdomen to directly visualize the externalsurfaces of the ovary, fallopian tubes and uterus to visualize any grosspathology which was not detected by previous examinations.

A high percentage of women who are unable to carry a pregnancy to fullterm undergo spontaneous abortion generally within the first six weeks.Pregnancy loss during the first six weeks has been shown to be as highas between 15 and 20%. Furthermore, the chance of a successful livebirth after consecutive abortions without a live birth is only 40-50%.

In vitro fertilization (IVF) requires the removal of ova from amammalian ovary, and exposure of these ova to sperm outside the body.Fertilization of each ovum requires that at least one living spermpenetrates the zona pellucida (outer covering) of the ovum and fuseswith the pronucleus. Once this has occurred and the ova are fertilized,they can be transferred to a uterus where they can become implanted onthe uterine wall. If implantation occurs, the pregnancy can proceed asif fertilization had occurred within the body. In vitro fertilizationhas gained widespread professional and public acceptance. However,despite the ever increasing frequency and refinement of this procedure,in vitro fertilization attempts most often do not result in pregnancy.In vitro pregnancy rates are currently only about 15 to 20 percent. Fora variety of reasons, exposing the ova to sperm does not necessarilyresult in fertilization. Furthermore, even where the ova is fertilized,the placement of the ova in a uterus usually does not result in normalimplantation. The low success rate of IVF often leads to an excessivefinancial and psychological burden for the infertile couple.

Other assisted reproductive technologies include two modifications ofthe IVF technique. The first is gamete intra-fallopian transfer (GIFT),the second is zygote intra-fallopian transfer (ZIFT). In the GIFTprocedure, the retrieved oocyte and sperm are mixed together and placedback into the fallopian tube where fertilization takes place. Thefertilized zygote then travels down through the fallopian tube into theendometrial cavity, where implantation may or may not take place. TheZIFT procedure allows for fertilization to take place in vitro as instandard IVF, and then the fertilized zygote is placed back in thefallopian tube where it then travels down into the uterus to implant.Finally, it is becoming realized that the hyper-stimulation protocolsnecessary to retrieve many oocytes from the donor woman may havedeleterious effects on the endometrium itself and decrease the rates ofimplantation. Two basic procedures have been utilized to help overcomethis problem. The first is considered non-stimulated oocyte retrieval. Asingle egg is retrieved, allowed to be fertilized and placed back intothe fallopian tube or uterus for implantation. The other techniqueinvolves the hyper-stimulation portion of the IVF procedure to retrievethe eggs and allow for fertilization in vitro. The zygotes are thenfrozen to be placed back into the patient after several normal cycles,with the hope that the endometrium will be more receptive toimplantation. All of these techniques attempt to maximize the quality ofthe eggs, zygotes produced after fertilization and the receptivity ofthe endometrium. Any procedure which would enhance the implantation rateabove the standard 15 to 20% would have a marked positive effect on anyof these technologies.

It is thus apparent that methods for improving the success rate ofassisted reproductive techniques in mammals are greatly desired. Meansfor determining the competence of particular fertilized ova,conceptuses, toward uterine implantation is particularly desired sincesuch means would lead to immediate improvement in the success rate ofassisted reproduction. Methods for improving the competence ofconceptuses toward implantation is likewise greatly desired.Additionally, methods for determining female infertility are alsodesired.

Contragestion, or post-coital contraception is currently practiced bytwo basic methods: surgical and medical. In the 1970's the "morningafter pill" (diethylstilbestrol) was popular as a post-coitalcontraceptive method. More recently, the use of the anti-progesteroneRU-486 has gained wide acceptance in Europe to terminate pregnancy soonafter fertilization and implantation. During the first trimester, themost common technique to end a pregnancy is by surgical abortion.Surgical abortions generally involve cervical dilation and curettage orvacuum aspiration. Finally, after the first trimester, labor inducingmedications such as oxytocin and prostaglandins can be utilized toinduce premature delivery and thus the termination of pregnancy. Themedical techniques described above are known to have a number of adversereactions and potential complications. The surgical technique can leadto uterine rupture, hemorrhage, and infection.

In the United States, the commonly employed contraceptive techniquesinclude oral steroidal contraceptives, injected or implanted steroidalcontraceptives, intra-uterine devices, physical, chemical, orphysicochemical barrier techniques, withdrawal, sexual abstinence aroundthe time of ovulation, breast feeding, and permanent sterilization. Inaddition to the high failure rates of some of these methods, a number ofthese methods have serious potential complications for the users. Forexample, in addition to metabolic changes induced by oralcontraceptives, there is possibly an increased risk of neoplasia,nutritional disorders, cardiovascular effects, thromboembolism and evendeath.

Methods for effecting contraception and contragestion are greatlydesired, especially methods which will exhibit low or no side effectstoward the patient. Methods which will inhibit contragestion at an earlystage in the chain of reproductive events are particularly desired andhave long been sought by persons skilled in reproductive science.

SUMMARY OF THE INVENTION

This invention provides methods of determining competence of a conceptustoward uterine implantation comprising administering transforming growthfactor β (TGFβ) to the conceptus and evaluating the level of productionby the conceptus of trophoblast fibronectin. Trophoblast fibronectinproduction is indicative of competence. Applicants have recognized thatembryo responsiveness to TGFβ is related to its overall liklihood ofimplantation, thus aiding in the selection of optimal embryos forimplantation.

The invention also provides methods of determining female infertility ina patient suspected of infertility comprising assaying the tissue orbodily fluid of the patient for the presence of transforming growthfactor β. Thus, the methods of this invention provide a tool fordiagnosing mammals with infertility due to inadequate TGFβ.

The invention further provides methods of increasing the success rate ofassisted reproduction comprising administering transforming growthfactor β to ovum, sperm or conceptus prior to, simultaneously with, orfollowing introduction of ovum, sperm or conceptus into the reproductivetract of a female mammal. Applicants have discovered that during normalmammalian pregnancy, trophoblast fibronectin, localized in theplacental-uterine junction, is important to implantation. Thus, TGFβ,which has been found to (1) concominantly stimulate the production oftrophoblast fibronectin; and (2) promote adhesiveness of trophoblast tothe extracellular matrix, effectively enhances the implantation of theovum or conceptus.

The invention still further provides methods of augmenting trophoblastfibronectin synthesis in a mammal comprising administering to the mammalan effective amount of transforming growth factor β. Applicants haverecognized the importance of trophoblast fibronectin in mammalianreproduction, and have discovered that augmenting the production oftrophoblast fibronectin is an important method of fertility therapy.Such augmentation has been found to be effected by transforming growthfactor β.

The invention also provides methods of inhibiting transforming growthfactor β synthesis in a mammal comprising administering a transforminggrowth factor β inhibitor, such as antisense oligonucleotides to mRNAcoding for transforming growth factor β, to the mammal. The inhibitorinterferes with the production or action of the transforming growthfactor β in the mammal. Inhibiting the production or action of TGFβ bythe methods of this invention provides inter alia, methods ofcontraception and contragestion.

The invention still further provides methods of inhibiting trophoblastfibronectin, especially tropho-uteronectin, TUN, synthesis in a mammalcomprising administering a transforming growth factor β antagonist tothe mammal in an amount effective to inhibit the production or effect ofthe growth factor in the mammal.

The invention also provides methods of inhibiting trophoblastfibronectin, especially tropho-uteronectin, TUN, synthesis in a mammalcomprising administering a transforming growth factor β receptorantagonist to the mammal in an amount effective to inhibit theproduction or effect of the growth factor in the mammal.

The invention further provides methods of contraception andcontragestion which comprise administering to a mammal a transforminggrowth factor β antagonist, such as antibodies to transforming growthfactor β, in an amount effective to increase the probability thatconception will be prevented in said mammal. TGFβ antagonists decreasethe amount of TGFβ available to stimulate, for example, TUN synthesis.This consequently renders a pregnancy unable to sustain itself and makesconception unlikely.

DETAILED DESCRIPTION OF THE INVENTION

Recently applicants have found that trophoblast fibronectins, especiallytropho-uteronectin (TUN), are synthesized by trophoblasts throughoutpregnancy at sites of attachment, both in vivo and in vitro.Tropho-uteronectin has been localized to the placental-uterine junction.It is believed that trophoblast fibronectins, and especiallytropho-uteronectin, have a critical function in modulating trophoblastadhesion to the uterine extracellular matrix. Feinberg, et al., 1991,American Journal of Pathology, 138(3): 537-543. In addition, it has beenestablished for many years that trophoblast cells of the conceptusestablish contact with the uterus as a critical part of the implantationprocess. Hertig, A. T. and Rock, J., 1956, American Journal of Anatomy,98, 435-494.

Applicants have now found that transforming growth factor β, (TGFβ),stimulates the production of trophoblast fibronectin includingtropho-uteronectin. Transforming growth factor β (TGFβ) as used hereinis a protein released from α-granules of platelets. TGFβ has recentlybeen localized at the human placental-uterine interface of implantationsites surgically removed from pregnant humans. Graham, et al., 1992,Biol. Reprod., 46: 561-572. TGFβ is available commercially such as fromSigma, St. Louis, Mo., R & D Systems, Minneapolis, Minn., andCollaborative Research, New Bedford, Mass. TGFβ refers to all of theisoforms of TGFβ. Thus, TGFβ1, TGFβ2, TGFβ3 and TGFβ4 may be encompassedby some or all aspects of the present invention.

Trophoblast fibronectin includes any and all of the fibronectin proteinsproduced by trophoblasts. One trophoblast fibronectin,tropho-uteronectin (TUN), has been found to be particularly important tothe practice of the present invention, however other trophoblastfibronectins are also believed to be important.

In accordance with the invention methods are provided for determiningthe competence of a conceptus toward uterine implantation comprisingadministering transforming growth factor β to the conceptus andevaluating the level of production by the conceptus of trophoblastfibronectin.

By the term competence toward uterine implantation is meantcharacteristics important to implantation. For example, the productionof fibronectin by trophoblasts is important to implantation. Thus, it isbelieved that the ability to elicit such response in vitro is anindication that the conceptus will effectively produce fibronectin andother factors important to development of the fetus in vivo followingits introduction into the uterus.

The term "conceptus" as used herein refers to the sum of derivatives ofa fertilized ovum at any stage of development from fertilization tobirth, including extraembryonic membranes, placenta, and trophoblasts,as well as the embryo or fetus. The methods of the present invention areapplicable to mammals generally. For example, methods of the presentinvention may apply, inter alia, to bovine, equine, porcine, canine,feline and human mammals.

The level of trophoblast fibronectin produced by the conceptus can beevaluated by any method which detects the protein, but which maintainsthe integrity of the conceptus. Thus, evaluation may be accomplished bycontacting the conceptus or the culture media surrounding the conceptuswith detectably labeled antibody specific for trophoblast fibronectin.Applicants have previously demonstrated ability of cultured trophoblaststo secrete fibronectin, and more specifically TUN, into the culturemedia. Feinberg, etal., 1991, American J. Pathology, 138(3): 537-543.For example, FDC-6 is a suitable antibody which recognizes trophoblastfibronectins such as trophouteronectin, as disclosed in U.S. Pat. No.4,894,326, incorporated by reference herein in its entirety. As oneskilled in the art will appreciate, other antibodies which specificallyrecognize one or more trophoblast fibronectins may also be used.

The detectable label is conveniently selected from the group consistingof enzymes, chromophores, fluorophores, coenzymes, chemiluminescentmaterials, enzyme inhibitors and paramagnetic metals andradionucleotides.

An assay also expected to be suitable for use in the present inventionis an in situ hybridization assay comprising the steps of contacting theconceptus with a detectably labeled oligonucleotide or cDNA probehybridizable with mRNA coding for trophoblast fibronectin, and detectingthe labeled oligonucleotide. General procedures for in situhybridization are as described for example in Stroop, et al., 1984, Lab.Invest. 51: 27-38 which reference is incorporated by reference herein inits entirety.

The methods of this invention may also be useful to determine femaleinfertility in a female mammal suspected of being infertile.Accordingly, tissue or bodily fluid of a patient may be assayed for thepresence of active and/or immunologic transforming growth factor β equalto the level of a fertile control. The presence of transforming growthfactor β is indicative of fertility. The lack of transforming growthfactor beta is indicative of lack of receptivity to implantation andconsequently, infertility. Bodily fluids expected to be useful include,e.g., plasma, serum and cervicouterine aspirates. Examples of cell typesexpected to be useful in such assays include an endometrial biopsy.Generally any reproductive bodily fluid or cell type associated withimplantation and the ability to stimulate synthesis of trophoblastfibronectin in a fertile control are expected to be useful.Conveniently, assays for the immunologically reactive quantity andactivity of functional TGFβ are commercially available and are easilyutilized by those skilled in the art.

In another aspect of this invention, methods of increasing the successrate of assisted reproduction are provided. These methods compriseadministering transforming growth factor β in vitro to a conceptus priorto introduction of said conceptus into the reproductive tract of afemale mammal. Transforming growth factor β is typically administered indoses of about 0.1 ng/ml to about 10 ng/ml. Preferably from about 0.5ng/ml to about 5 ng/ml is administered. Still more preferably, fromabout 1 ng/ml to about 3 ng/ml of TGFβ is administered, theconcentrations referring to the fluid in which the conceptus issuspended. In other preferred embodiments of the present invention fromabout 1.5 ng/ml to about 2.5 ng/ml TGFβ are administered to a conceptus.Administration can be, for example, by addition of TGFβ to the culturemedium. In such case the concentrations of TGFβ refer to the finalconcentration in the fluid environment of the conceptus.

In another aspect of this invention, a method of augmenting trophoblastfibronectin production in a mammal is provided comprising administeringto the mammal an effective amount of TGFβ. Administration may beaccomplished by any method known to those skilled in the art. Forexample, TGFβ may be administered by interuterine infusion, gels, orphysiological solutions. Administration may also be accomplishedsystemically such as parenterally, intravenously, subcutaneously, orintradermally. For example, a "patch" which delivers TGFβ intradermallymay be worn in the pubis area.

TGFβ may be administered by any one of these methods prior to theintroduction of ovum, sperm, or conceptus into the reproductive tract ofa female mammal, either naturally or by assisted reproductivetechniques. For example, an interuterine infusion, gel or physiologicalsolution containing TGFβ may be used to introduce TGFβ into the vagina,cervical canal, uterus, and fallopian tubes. Furthermore, in cases ofassisted reproductive techniques, TGFβ may be contacted with ovum orconceptus in vitro prior to introduction into the reproductive tract.

TGFβ may also be administered simultaneously with the introduction ofovum, sperm or conceptus into the reproductive tract of a female mammal.For example, a gel may be prepared containing TGFβ in which a conceptusmay be suspended during in vitro fertilization, ovum and sperm may besuspended during gamete intra-fallopian transfer or zygote may besuspending during intra-fallopian transfer. Physiological solutionscontaining TGFβ may also be administered contemporaneously with assistedreproductive procedures such as these.

In accordance with still other methods of the present invention, TGFβmay be administered by these methods following introduction of ovum,sperm or conceptus into the reproductive tract of a female mammal. Forexample, an intravenous injection of a physiological solution of TGFβfollowing introduction of an ovum, sperm, or conceptus into the uterusmay be administered as a precautionary procedure to bolster the chancesthat a pregnancy will be sustained. Of course, one skilled in the artwill appreciate that dosage and methods of administration will vary withthe size, weight, and conditions of the patient being treated, the goalbeing to increase trophoblast fibronectin synthesis to levels of anormal fertile control.

In still another aspect of this invention, methods of inhibitingtransforming growth factor β synthesis in a mammal are providedcomprising administering a transforming growth factor β inhibitor tosaid mammal in an amount effective to inhibit transforming growth factorβ synthesis in said mammal. For example, antisense oligonucleotides canbe used to inhibit TGFβ synthesis by mammalian trophoblasts orendometrium. Recently it has been demonstrated that addingoligonucleotide antisense DNA probes to cells causes them tospecifically stop producing the corresponding protein. See, e.g.,Tortora; et al. (1990), Proc. Natl. Acad. Sci. U.S.A. 87, 705-708. Anantisense oligonucleotide can be readily made and administered in anumber of ways known to those skilled in the art. See, for example, U.S.Pat. No. 5,098,890 issued March 24, 1992.

Inhibition of TGFβ synthesis in a mammal has a variety of utilities. Forexample, a mammal determined to have a level of TGFβ equal to or inexcess of a normal fertile control may be a candidate for TGFβinhibition whereby inhibition is designed to bring levels of TGFβ withinthe range of a normal fertile control. TGFβ inhibition can also beemployed to maintain a deficient level of TGFβ concentration as comparedto a normal fertile control as a method of contraception. Additionally,TGFβ inhibition to below the level of TGFβ present in a normal fertilefemale can be utilized to terminate a pregnancy and thus provide amethod of contragestion.

In another aspect of this invention, methods of inhibiting trophoblastfibronectin synthesis in a mammal below the level of trophoblastfibronectin found in a normal fertile female is provided comprisingadministering a transforming growth factor β antagonist or TGFβ receptorantagonist to said mammal in an amount effective to inhibit trophoblastfibronectin synthesis in said mammal. For example, antibodies againstTGFβ or TGFβ receptors may be administered to said mammal. Suchantibodies can be prepared by standard methods known to those skilled inthe art. Alternatively, such antibodies are available commercially suchas from R & D Systems, Minneapolis, Minn., and may be administered bywell known methods.

Immunologic interruption of pregnancy can be achieved. For example, ithas been shown that when 5 and 25 mg of purified anti-hCG was injectedinto three patients with ectopic pregnancies, one of the patientscompletely resolved her tubal pregnancy, while the two others hadmarkedly decreased levels of progesterone and estrogen, suggesting amarked decrease in viability of the pregnancy. Frydman et al., "Phase Iclinical trial of monoclonal anti-human chorionic gonadotropin antibodyin women with an ectopic pregnancy," Fertil Steril 52: 734-8 (1989).These authors used mouse monoclonal antibodies. In a more recent articleusing human monoclonal antibodies, it was shown that humanizedantibodies could be utilized in the treatment of CMV after renaltransplantation. Skarp et al., "Use of a human monoclonalanti-cytomegalovirus antibody for the treatment of severecytomegalovirus after renal transplantation," Transplant Proc 22: 234(1990).

In addition to being given systemically, these particular monoclonalantibodies can also be applied directly within the intrauterine cavityand possibly within the fallopian tube as described above.

Administration of TGFβ and inhibitors and antagonists thereof can beaccomplished as described above, for example, parenterally, byintravenous injection, by interuterine infusionsβ, gels, or sponges orin other ways apparent to persons of skill in the art. Literature isknown describing use of these methods for treatment of a variety ofconditions. It has been shown that the endocrine function of an ovarycan be markedly changed by an intrauterine infusion. Helmer, et al.,1989, J. Reprod. Fertil., 87: 89-101. It has been shown that rat uteriwhich received an intrauterine injection of luteinizing releasinghormone had a significantly increased rate of implantation compared touteri which had no injection. Jones, R. C. "Blastocyst attachment in theovariectomized rat treated with an intrauterine injection of luteinizinghormone-releasing hormone (LRH)," Acta Endocrinol (Copenh) 103: 266-8(1983). In addition to the use of solutions, the use of gels which areinstilled intracervically to facilitate labor and delivery is known. Seee.g., Ekman et al., "Intracervical instillation of PGE2-gel in patientswith missed abortion or intrauterine fetal death," Arch Gynecol 233:241-5 (1983). Additionally, an intrauterine vehicle either similar tothose currently existing on the market or modified to facilitate slowerrelease of a pharmacologic agent which might either enhance or decreasethe synthesis of TGFβ or TUN can be utilized. An example of such a slowrelease intrauterine vehicle can be found in Zhu et al. "The effect ofintrauterine devices, the stainless steel ring, the copper T220, andreleasing levonorgestrel, on the bleeding profile and the morphologicalstructure of the human endometrium--a comparative study of three IUDs. Amorphometric study of 96 cases," Contraception 40: 425-38 (1989).

Kits for determining fibronectin production are also provided inaccordance with the present invention comprising transforming growthfactor β in a physiologically acceptable solution and an assay fortrophoblast fibronectin. Conventional kit components such as bufferingagents, antibacterial agents, stabilizing agents and excipient are alsoencompassed in kits of the present invention. Such components are wellknown in the art and are discussed, for example, in The United Statespharmacopeia--The National Formulary, 22nd Revision, Jan. 1, 1990, MackPublishing Company, Easton, Pa., Remington's Pharmaceutical Sciences,Gennaro, A. R., ed., Mack Publishing Company, Easton, Pa. (1985), thedisclosures of each of which are hereby incorporated herein by referencein their entirety.

The following examples are merely illustrative of the present inventionand should not be considered as limiting the scope of the invention inany way. These examples and equivalents thereof will become moreapparent to those versed in the art in light of the present disclosure,and the accompanying claims.

EXAMPLE 1

Cell Culture

Cytotrophoblasts were prepared from a normal term placenta by the methodof Kliman et al., 1986, Endocrinology, 118: 1567-1582. Isolatedtrophoblasts were counted as described in Kliman, supra. Prior to cellplating, trophoblasts were suspended in Dulbecco's minimal essentialmedia (DMEM) with added glutamine and gentamicin, with or without serum.The concentration of this cell suspension prior to plating was 2×10⁶/ml.

EXAMPLE 2

Identification of a Platelet Derived TUN Stimulating Factor in Serum

On glass or plastic substrates, trophoblast cell cultures were preparedas described in Example 1. The production of TUN by trophoblasts inserum-containing medium was observed by culturing trophoblasts indifferent concentrations of serum. The production of TUN was determinedto be dose-dependent, with concentrations of detectable TUN in the mediaincreasing stepwise from 1 to 20 μg/ml as the amount of serum in themedia was increased stepwise from 1 to 10%. One percent to 10%serum-containing media produced cells which appeared morphologicallyidentical, with good evidence of spreading and formation of aggregatesand syncytia. No TUN production was observed when trophoblasts arecultured in serum-free media and in the absense of TGFβ.

The use of a cord serum sample from a baby with severe alloimmunethrombocytopenia (severe lack of platelets) resulted in very little TUNstimulation, suggesting that a critical TUN stimulating factor isplatelet derived.

To verify that TUN stimulating factor was in fact derived fromplatelets, blood was drawn from a healthy donor, and separated into twocentrifuge tubes with anti-coagulant added. One tube was spun at highspeed (2000 rpm×10 min). The other tube was spun at low speed (500rpm×10 min). The high speed tube had no platelets in the supernatant andthe low speed tube had virtually all platelets remaining in thesupernatant. Both plasmas were induced to clot and the resultant serumwere labeled as platelet rich (low speed spin) and platelet poor (highspeed spin). Only the platelet rich serum induced trophoblasts to makeincreasing amounts of TUN.

EXAMPLE 3

Stimulation of TUN by Addition of TGFβ

Trophoblasts prepared as described in Example 1 were cultured in 2%platelet poor serum or in serum derived from the alloimmunethrombocytopenic neonate in the presence of exogenously added TGFβ1. 50and 200 pM TGFβ elicited a response of 3 to 4 fold induction of TUNafter 48 hours, with levels of TUN in the media increasing fromapproximately 1 μg/ml to 4 μg/ml.

EXAMPLE 4

TGFβ Antagonist Inhibits Production of TUN

Platelet-rich serum was preincubated separately for 6 hours with twodifferent commercially available TGFβ neutralizing antibodies (R & DSystems, Minneapolis, Minn.) at concentrations of 50 to 100 μg/ml.Trophoblasts prepared as described in Example 1 using serum preincubatedwith the TGFβ neutralizing antibodies exhibited undetectable levels ofTUN synthesis by Western imnunoblots. (Addition of 1 ng/mlplatelet-derived growth factor to trophoblast cultures for 48 hourseither alone or in combination with 1 ng/ml TGFβ, had no additive effecton TUN production). This finding further confirms that TGFβ has asignificant role in the stimulation of TUN.

EXAMPLE 5

Preparation of Plates

Six-well plastic dishes were precoated with a solution of plasmafibronectin (Boehringer) prepared at a concentration of 10 μg/ml inphosphate buffered saline. One ml of this solution was applied to eachsix well dish. The plates were incubated at room temperature for 8 to 10hours.

EXAMPLE 6

Effect of added TGFβ on trophoblast attachment

The effect of TGFβ on trophoblast attachment to plasma fibronectinsurfaces was examined. One ml of cell suspension in serum-free mediaprepared as described in Example 1 was added to each dish of a six welldish prepared as described in Example 5. Following plating of the cells,a stock solution (1 ng/μl) of transforming growth factor β (R & DSystems, Minneapolis, Minn.) was added to the cell culture giving afinal concentration of 1 ng/ml in the trophoblast cultures whichreceived TGFβ.

Cells were cultured for 48 hours. Thereafter medium was removed, thecultures were washed gently with PBS and fixed with 10% neutral bufferedformalin for 10 minutes. Detailed examination of the cells by lightmicroscopy revealed a clear quantitative difference between cellstreated with TGFβ and those not treated with TGFβ. In the absence ofpre-coated plasma fibronectin, about 97% of the cells were round. Withadded plasma fibronectin, coated at 10 μg/ml, about 70% of the cellswere round, the remainder divided between intermediate and flat. Withthe addition of 1 ng/ml of acid activated TGFβ, less than 25% of thecells were round, almost 40% were intermediate, and about 35% of thecells were flat. These results suggest that the combination of bothpre-coated plasma fibronectin, and added TGFβ, which stimulatestrophoblast secretion of TUN, are capable of enhancing trophoblastattachment to culture surfaces under serum free conditions.

Further, the pre-coated fibronectin was found to be easily degraded bythe trophoblasts, releasing into the media several proteolyticfragments. Conversely, pre-coated amniotic fluid and trophoblastfibronectin were very resistant to digestion by trophoblasts and scantlevels of proteolytic fragments were found. This may explain howtrophoblasts can simultaneously invade and digest the maternal uterineextracellular matrix, yet synthesize and deposit new TUN-containing,protease resistant extracellular matrix components during implantation.

What is claimed is:
 1. A method of determining uterine implantationcompetence of a conceptus prior to implantation comprising:obtaining atrophoblast sample from the conceptus prior to uterine implantation ofthe conceptus; administering transforming growth factor β to thetrophoblast sample; and evaluating production by the trophoblast sampleof trophoblast fibronectin; trophoblast fibronectin production by thetransforming growth factor β-administered trophoblast sample beingindicative of uterine implantation competence of the conceptus.
 2. Themethod of claim 1 wherein trophoblast fibronectin is tropho-uteronectin.3. The method of claim 1 wherein the step of evaluating comprisescontacting the transforming, growth factor β-administered trophoblastsample or culture media obtained from cultures containing thetransforming growth factor β-administered trophoblast sample with adelectably labeled antibody specific for trophoblast fibronectin anddetecting the specific binding of the labeled antibody to trophoblastfibronectin present in the trophoblast sample or the culture media. 4.The method of claim 3 wherein the antibody is anti-trophouteronectinantibody FDC-6.